Genetic predisposition is the primary risk factor for familial breast cancer. For the majority of familial breast cancer, however, the genetic predispositions remain unknown. All newly identified predispositions occur rarely in disease population, and the unknown genetic predispositions are estimated to reach up to total thousands.
Wen et al BMC Cancer 2014, 14:470 http://www.biomedcentral.com/1471-2407/14/470 RESEARCH ARTICLE Open Access Family-specific, novel, deleterious germline variants provide a rich resource to identify genetic predispositions for BRCAx familial breast cancer Hongxiu Wen1†, Yeong C Kim1†, Carrie Snyder2, Fengxia Xiao1, Elizabeth A Fleissner3, Dina Becirovic2, Jiangtao Luo4, Bradley Downs1, Simon Sherman3, Kenneth H Cowan3, Henry T Lynch1,2,5* and San Ming Wang1,3* Abstract Background: Genetic predisposition is the primary risk factor for familial breast cancer For the majority of familial breast cancer, however, the genetic predispositions remain unknown All newly identified predispositions occur rarely in disease population, and the unknown genetic predispositions are estimated to reach up to total thousands Family unit is the basic structure of genetics Because it is an autosomal dominant disease, individuals with a history of familial breast cancer must carry the same genetic predisposition across generations Therefore, focusing on the cases in lineages of familial breast cancer, rather than pooled cases in disease population, is expected to provide high probability to identify the genetic predisposition for each family Methods: In this study, we tested genetic predispositions by analyzing the family-specific variants in familial breast cancer Using exome sequencing, we analyzed three families and 22 probands with BRCAx (BRCA-negative) familial breast cancer Results: We observed the presence of family-specific, novel, deleterious germline variants in each family Of the germline variants identified, many were shared between the disease-affected family members of the same family but not found in different families, which have their own specific variants Certain variants are putative deleterious genetic predispositions damaging functionally important genes involved in DNA replication and damaging repair, tumor suppression, signal transduction, and phosphorylation Conclusions: Our study demonstrates that the predispositions for many BRCAx familial breast cancer families can lie in each disease family The application of a family-focused approach has the potential to detect many new predispositions Background Breast cancer is a leading cancer in women [1] About 10-20% of breast cancer cases are family clustered, with multiple family members affected by the disease [2] Genetic predispositions are the major risk factor for the * Correspondence: htlynch@creighton.edu; sanming.wang@unmc.edu † Equal contributors Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA Fred & Pamela Buffett Cancer Center, Omaha, USA Full list of author information is available at the end of the article disease However, the genetic predispositions are currently known for only 30-40% of the familial breast cancer disease families The remaining 60-70% of women with familial breast cancer have unknown predispositions and are diagnosed with BRCAx, for their unknown predisposition of familial breast cancer [3] It is estimated the “missing” heredity trait for BRCAx families likely consists of thousands of rare variants, each presenting a minor disease risk [4] Indeed, broadly screening the variants across disease populations has uncovered multiple new genetic predispositions for familial breast cancer A consistent pattern among these newly classified predispositions is that © 2014 Wen et al.; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Wen et al BMC Cancer 2014, 14:470 http://www.biomedcentral.com/1471-2407/14/470 they are always present at very-low frequencies in the given disease population [5-10] Their extreme rarity implies that a greater sampling size of disease populations is required to identify the germline predispositions [10] However, such an expansion is deemed to increase the complexity of data analysis, experimental costs, and time needed As such, focusing only on the rare variants will not likely be able to determine the entire spectrum of genetic predispositions for BRCAx familial breast cancer families New alternative hypotheses and approaches must be explored to improve the situation For example, mosaic mutation has implications as potential predispositions for familial breast cancer [11] Familial breast cancer is defined as an autosomal dominant genetic disease [12] Although incidences of breast cancer often exhibit atypical Mendelian patterns due to the factors such as low penetrance of genetic predispositions, the predisposition in a disease-prone family is expected to transmit across generations and shared between family members Focusing on each disease family with a history of the disease is expected to improve the chance to detect the predisposition in a family compared to screening the disease population of pooled cases without family relationships, which can dilute the predisposition highly prevalent in a disease family into insignificant level We hypothesize that the unknown predispositions for many BRCAx familial breast cancer are specific to each family with a history of the disease Our previous exome study of a BRCAx familial breast cancer family shows the presence of rich genetic variants [13] In the present study, we expand the exome sequencing study by analyzing three families with BRCAx familial breast cancer; 17 members had cancer, and five members were without cancer Our study also includes 22 probands of BRCAx familial breast cancer Our study reveals the presence of family-specific, novel, deleterious genetic variants as putative genetic predispositions in each family with BRCAx familial breast cancer Methods Use of human subjects The use of the patient samples for the study was approved by the Institutional Review Boards (IRB) of Creighton University School of Medicine (#00-12265 ) and University of Nebraska Medical Center (718-11-EP) All subjects signed the Consent to Participate Form for cancer genetic study Individuals from three families with BRCAx breast cancer were used to generate exome sequences as we have previously described [13] Family I included six individuals with breast cancer and two individuals without breast cancer Family II included five individuals with breast cancer, one obligate carrier and two Page of 11 individuals without breast cancer Family III included five individuals with breast cancer and one individual without breast cancer Additionally, 22 probands for BRCAx familial breast cancer were included in exome sequencing All cases used in the study were BRCA1negative, and BRCA2-negative, 41 were female and were male, the average age is 42 years old (Figure 1, Table 1) Exome sequencing For each sample, exome sequencing used DNA from blood cells Exome libraries were constructed using the TruSeq Exome Enrichment Kit (62 Mb, Illumina, San Diego, CA) as per manufacturer’s procedures Exome sequences were collected with a HiSeq™ 2000 sequencer (Illumina, San Diego, CA) with paired-end (2 × 100) All exome data were deposited in the Sequence Read Archive (SRA) database in the National Center for Biotechnology Information (NCBI) (Accession numbers SAMN02404413- SAMN02404456) Exome sequence mapping and variant calling Exome sequences were mapped to the human genome reference sequence hg19 by Bowtie2 with default parameters in paired mode [14] The subsequent SAM files were converted to BAM files Duplicates were removed using Picard (http://picard.sourceforge.net) The mapped reads were locally realigned using the genome mapping tool RealignerTargetCreator from the Genome Atlas Tool Kit (GATK) [15] The base quality scores were recalibrated using BaseRecalibrator (GATK), with NCBI dbSNP build 137, in the GATK resource bundles for reference sequence hg19 VarScan was used for variant calling, [16] VarScan was run on pileup data generated from BAM files using SAMtools utilities [17] The mpileup command, with –B parameter to disable base alignment quality (BAQ) computation, and the default parameters were used, with the minimum read depth at 10 and the minimum base quality at 30 The called variants were annotated with ANNOVAR using the software-provided databases of the Reference Sequence (RefSeq; NCBI), dbSNP 137, the 1000 Genomes Project, and the NIH Heart, Lung and Blood Institute (NHLBI) Exome Sequencing Project (ESP) 6500 (http:// evs.gs.washington.edu) Those that matched in the databases were classified as known variants and removed Family-specific normal variants were eliminated by removing the variants shared between the affected and the unaffected family members in each family The remaining novel variants were classified into synonymous, non-synonymous, splicing site change, stop gain- or loss groups The variants causing synonymous changes were then removed For the remaining variants, PolyPhen-2 was used to identify variants causing deleterious effects in the affected genes [probably damaging score: 0.909-1; possibly damaging Wen et al BMC Cancer 2014, 14:470 http://www.biomedcentral.com/1471-2407/14/470 Page of 11 Family 1 Pro Br Br Pro Br Pro Pro Br Bl Bl Lu Br Bt Br Bt Br Br Family Lym Br Br Br Br Br NHL Family 3 Co Lu Br Br En Br Br Ki Pro Co Cx Br Sar Sk Br Br Br Br Figure Pedigrees of the three families used in the study BC (breast cancer), Bt (brain tumor), CRC (colorectal cancer), Lu (lung cancer), En (endometrium cancer), Ki (kidney cancer), Lym (lymphoma), NHL (non-Hodgkin lymphoma), OC (ovarian cancer), Pro (prostate cancer) Sar (sarcoma), Sk (skin cancer) score: 0.447 - 0.908; Benign score: - 0.446; HumVar score: 18] The variants defined as benign were removed These processes generated a list of novel, deleterious variants only present in the cancer-affected family members and probands, Note that the variants in probands were filtered by population databases only Power calculation Using a two-sided paired t-test and assuming a genetic relative risk (GRR) equal to 5.8, disease prevalence equal to 0.03, a disease locus frequency equal to 0.01, and a sib recurrence ratio of 2, a sample size of 20 achieves 81% power to detect a mutation difference with a (standardized) effect size of 0.67 between the affected member and the unaffected member The significance level (alpha) is, in turn, 0.05 [19,20] Validation Sanger sequencing was used to validate deleterious variants Sense and antisense PCR primers for each selected variant were designed using the Primer3 program The original DNA samples that were used in exome sequencing were served as PCR templates PCR amplicons were subjected to BigDye sequencing The resulting sequences were evaluated using CLC Genomics Workbench Program (Cambridge, MA) to confirm the variants called from exome sequences Results Mapping exome data and calling variants Exome sequences were collected via a blood sample from each study participant and mapped to the human genome reference sequence hg19 Variants were called from the mapping data We focused on single-base, non- Wen et al BMC Cancer 2014, 14:470 http://www.biomedcentral.com/1471-2407/14/470 Page of 11 Table BRCAx familial breast cancer cases used in the study Family Cancer type Pathology BRCA1/2 Exome Reads Bases Bases map rate (%) Coverage Variant called Family 1 Breast Infiltrating ductal - 42,973,730 4,340,346,730 97.6 70 184,865 Breast Not available - 40,158,059 4,055,963,959 98.3 65 152,692 Breast Infiltrating ductal - 46,240,754 4,670,316,154 97.2 75 176,554 Prostate Adenocarcinoma - 23,418,595 2,365,278,095 98.1 38 207,103 - 40,313,161 4,071,629,261 98.0 66 213,347 Adenocarcinoma - 17,496,012 1,767,097,212 97.9 28 183,741 No Cancer Breast, Colon Brain Not available - 36,166,319 3,652,798,219 99.5 59 171,425 Breast Adenocarcinoma - 27,830,687 2,810,899,387 96.3 45 104,343 Breast, Breast Medullary, infiltrating ductal - 33,419,098 3,375,328,898 92.9 54 113,079 Family 2 Obligated carrier Breast Infiltrating ductal - 27,261,117 2,753,372,817 92.4 44 115,328 - 40,973,473 4,138,320,773 99.6 67 127,272 Breast Ductal carcinoma in situ - 29,561,523 2,985,713,823 91.5 48 108,655 Breast Infiltrating ductal - 25,790,969 2,604,887,869 93.1 42 84,687 Infiltrating ductal Breast - 37,657,589 3,803,416,489 91.6 61 139,891 No Cancer - 17,433,912 1,760,825,112 91.6 28 131,786 No Cancer - 35,977,512 3,633,728,712 97.3 59 128,680 Family Endometrial Adenocarcinoma - 33,662,978 3,399,960,778 93.2 55 129,754 Breast, Skin Basal, infiltrating ductal - 29,648,460 2,994,494,460 98.3 48 198,862 - 53,411,156 5,394,526,756 98.8 87 193,017 Infiltrating ductal - 31,736,845 3,205,421,345 98.3 52 130,941 No Cancer Breast Breast Ductal carcinoma in situ - 35,014,538 3,536,468,338 98.4 57 129,754 Breast Not available - 38,418,769 3,880,295,669 97.5 62 161,953 Breast Ductal carcinoma in situ - 17,832,681 1,801,100,781 93.1 29 109,864 Probands Breast Invasive ductal carcinoma - 36,166,319 3,652,798,219 99.5 59 142,155 Breast Invasive ductal carcinoma - 50,944,516 5,145,396,116 98.4 83 152,125 Breast Invasive ductal carcinoma - 43,889,986 4,432,888,586 99.6 71 169,633 Breast Invasive ductal carcinoma - 40,125,408 4,052,666,208 99.5 65 153,511 Breast Invasive lobular carcinoma - 31,798,628 3,211,661,428 97.5 52 119,875 Breast Invasive ductal carcinoma - 49,739,415 5,023,680,915 99.6 81 113,058 Breast Invasive ductal carcinoma - 63,352,269 6,398,579,169 99.6 103 99,732 Breast Invasive ductal carcinoma - 43,744,840 4,418,228,840 99.5 71 149,873 10 Breast Invasive ductal carcinoma - 43,573,311 4,400,904,411 99.6 71 141,236 11 Breast Invasive ductal carcinoma - 40,938,838 4,134,822,638 99.3 67 143,262 12 Breast Ductal carcinoma in situ - 36,258,870 3,662,145,870 99.6 59 138,018 13 Breast Ductal carcinoma in situ - 34,550,745 3,489,625,245 99.4 56 146,858 14 Breast Invasive ductal carcinoma - 50,295,200 5,079,815,200 99.5 82 156,666 15 Breast Invasive ductal carcinoma - 60,736,566 6,134,393,166 99.7 99 115,909 16 Breast Invasive ductal carcinoma - 57,383,360 5,795,719,360 99.6 93 120,945 Wen et al BMC Cancer 2014, 14:470 http://www.biomedcentral.com/1471-2407/14/470 Page of 11 Table BRCAx familial breast cancer cases used in the study (Continued) 17 Breast Invasive ductal carcinoma - 44,922,611 4,537,183,711 99.6 73 110,503 18 Breast Invasive ductal carcinoma - 33,883,509 3,422,234,409 99.4 55 131,955 19 Breast Invasive ductal carcinoma - 49,729,619 5,022,691,519 99.5 81 146,665 20 Breast Invasive ductal carcinoma - 63,184,143 6,381,598,443 99.6 103 119,680 21 Breast Invasive ductal carcinoma - 28,002,381 2,828,240,481 99.6 46 86,924 22 Breast Invasive ductal carcinoma - 47,794,798 4,827,274,598 99.5 78 112,030 38,941,211 3,933,062,277 97.7 Average synonymous variants that affect protein coding, splicing, and stop gain- or loss mutations, which are reliably detectable by exome analysis [21] The average exome coverage was 63x, and the average number of variants called was 140,187 per case (Table 1) To increase the likelihood that the variants identified in the breast cancer-affected family members are breast cancer-associated, variants in each data set were filtered by: 1) removal of common variants present in human populations All variants matching to population-derived variant databases (i.e., dbSNP137, ESP6500, and 1000 genomes) were removed; 2) Removal of family-specific normal variants For the three families in the study, the variants shared between the affected and the unaffected members in the same family were removed To identify those causing deleterious effects in the affected genes, the remaining variants were analyzed using the Polyphen-2 Program [18] A total of 337 novel, deleterious variants present only in the affected members of Families I, II, and III were identified at an average of 112 variants per family (Table 2, Additional files 1: Table S1A, B, C); 689 novel, deleterious variants were identified in the 22 probands at an average of 30 variants per proband (Table 2, Additional files 2: Table S2A, B) Sanger sequencing validated the mapped variants at a validation rate of 83% (53/64), highlighting the reliability of the variants identified by exome mapping analysis (Additional file 1: Table S1D) Novel deleterious variants are mostly family-specific We compared the variants within each family We observed that 25% of the variants on average (14% in Family I, 29% in Family II, 35% in Family III) were shared in multiple affected members in each family, whereas 75% on average (86% in Family I, 71% in Family II and 65% in Family III) were present only in single affected member in each family (Table 2) We then compared the shared variants between the three families, and found only variant was shared between Family I and Family II, four variants were shared between Family I and Family III (Figure 2A) For the 689 variants identified in the probands, 82% were proband-specific, and only 18% were shared between probands at various frequencies (Figure 2B, Additional file 2: Table S2A, S2B) 63 140,187 The results indicate that the majority of the novel, deleterious variants identified in the three families and probands are family-specific, i.e., present only in each family but not shared with other families Identification of putative genetic predispositions We analyzed the shared mutations between the affected members of the same family, the functional class of the mutated genes, and existing evidence for their contribution to cancer In doing so, we identified the variants as the putative predispositions in Family I, II, and III, and probands (Table 3, Additional file 1: Table S1A, S1B, S1C) For Family I, this was the PTEN-Induced Putative Kinase (PINK1); for Family II, these were Lysine (K) Acetyltransferase 6B (KAT6B) and Neurogenic Locus Notch Homolog Protein (NOTCH2); and for Family III, this was Phosphorylase Kinase Beta (PHKB) PINK1 is a mitochondrial serine/threonine-protein kinase Mutation in PINK1 causes autosomal recessive Parkinson’s disease [22] KAT6B is a histone acetyl transferase involved in DNA replication, gene expression and regulation, and epigenetic modification of chromosomal structure [23] Mutations in KAT6B cause multiple neurological diseases [24] NOTCH2 is a member of the Notch family involved in controlling cell fate decision Low Notch activity leads to hyperproliferative activity in breast cancer [25] and mutation in NOTCH2 causes HajduCheney syndrome [26] PHKB regulates the function of phosphorylase kinase [27] Mutation in PHKB causes glycogen storage disease type 9B [28] Interestingly, a variant in Polymerase (DNA-Directed) Kappa (POLK) was present in Family I member #4 POLK is a member of Y family DNA polymerases, and functions by repairing the replication fork passing through DNA lesions [29] Although we are not able to validate it due to the lack of DNA from the subject’s parents, it raises a possibility that this variant could be a de novo mutation in this individual Multiple transcriptional factors were also affected by the mutations in each family For example, the following transcriptional factors were mutated in Family I: ZNF335, LRRC66, ZNF417, ZNF587, GTF2I, ZFAND4, EIF4G2, GZF1, CCDC86, ZSCAN18, ZNF546, TAF1L, and LRIG3 (Additional file 1: Table S1A) Wen et al BMC Cancer 2014, 14:470 http://www.biomedcentral.com/1471-2407/14/470 Page of 11 Table Novel, deleterious variants detected in breast cancer-affected cases* Family Total (%) Individual (%) Shared**(%) 37 35 2 26 26 25 15 48 29 Table Novel, deleterious variants detected in breast cancer-affected cases* (Continued) 18 57 25 32 19 58 18 40 20 47 23 24 21 33 25 10 22 34 22 12 39 Total 689 (100) 568 (82) 121 (18) 17 12 Per proband 30 26 12 6 14 143 (199) 123 (86) 20 (14) 22 13 15 10 21 12 21 12 16 8 66 (100) 47 (71) 19 (29) 39 13 26 48 27 21 21 12 32 12 20 Family Subtotal Family Subtotal *The counts in subtotal and total are the unique number of variants **Shared with family members in the families, or shared with other probands The variant data from probands show similar patterns as those of the three families (Table 3) In the 22 probands, four carried variants affecting the genes involved in DNA replication and damaging repair Those include Polymerase (DNA-directed) Theta (POLQ) in Proband #2, RAD23 Homolog B (S cerevisiae) (RAD23B) in Proband #3, Ligase A Family 41 19 22 Subtotal 128 (100) 83 (65) 45 (35) Total 337 (100) 253 (75) 84 (25) 35 10 25 58 22 36 74 28 46 77 49 28 70 28 42 41 16 25 31 24 43 27 16 Probands 51 19 32 10 61 30 31 11 70 35 35 12 51 12 39 13 55 15 40 14 60 30 30 15 51 31 20 16 41 31 10 17 32 18 14 B Figure Comparison of the variants in BRCAx families and probands A Comparison in the three families B Comparison in the probands The results show that the variants detected in the cancer-affected family members are highly family-specific The higher rate (18%) of the shared variants in the probands are likely due to the remaining normal variants not filtered in the probands and the larger number of families represented by the probands than the three families Wen et al BMC Cancer 2014, 14:470 http://www.biomedcentral.com/1471-2407/14/470 Page of 11 Table Putative predispositions in familial breast cancer families and probands Gene Description Position Nucleotide Amino acid Type PolyPhen2* Cancer-affected member Frequency Score prediction Family 1 GPRIN1 G protein regulated inducer of neurite outgrowth chr5:176026123 c.T713C p.L238S PINK1 PTEN induced putative kinase chr1:20972051 c.960-2A > G POLK Polymerase (DNA directed) kappa chr5:74892737 c.A2219G p.H740R KAT6B K(lysine) acetyltransferase 6B chr10:76789128 KAT6B K(lysine) acetyltransferase 6B Exonic 0.91 D - + + + + + - Splicing NA NA - - + + - - - Exonic 0.62 P - - - - - c.G4546T p.D1516Y Exonic 0.95 D - + + + + + chr10:76789311 c.C4729T p.R1577C Exonic 0.96 D - + + + + + chr1:120459167 c.C6178T p.R2060C Exonic 0.99 D - - Family + - NOTCH2 Notch Family + - - + NANP N-acetylneuraminic acid phosphatase chr20:25596725 c.A583G PHKB phosphorylase kinase, beta chr16:47628126 c.1204 + 1G > T p.I195V Exonic 0.98 D + - Splicing NA NA - + - Proband JAKMIP3 Janus kinase and microtubule interacting protein chr10:133955524 c.G1574C p.G525A Exonic 1.00 D POLQ Polymerase (DNA directed), theta chr3:121207798 c.A3980C p.Q1327P Exonic 1.00 D DUX2 Double homeobox chr10:135494906 Splicing NA NA UBE2L3 Ubiquitin-conjugating enzyme E2L chr22:21975938 c.G349A p.E117K Exonic 0.96 D RAD23B RAD23 homolog B (S cerevisiae) chr9:110087260 c.C1028T p.P343L Exonic 0.99 D GATA3 GATA binding protein chr10:8100630 c.C604T p.R202C Exonic 0.92 D KAT6B K(lysine) acetyltransferase 6B chr10:76744854 c.G2390A p.S797N Exonic 0.98 D LIG1 Ligase I, DNA, ATP-dependent chr19:48637322 c.G1525A p.E509K Exonic 0.95 D 10 LIG4 Ligase IV, DNA, ATP-dependent chr13:108862463 c.G1154A p.R385K Exonic 1.00 D 14 NOTCH2 Notch chr1:120529603 c.G854A p.R285H Exonic 1.00 D 15 ABL1 c-abl oncogene 1, non-receptor tyrosine kinase chr9:133729493 c.G122A p.G41D Exonic 0.92 D 16 TNK2 Tyrosine kinase, non-receptor, chr3:195596385 c.C1760T p.P587L Exonic 1.00 D 17 NFRKB Nuclear factor related to kappaB binding protein chr11:129755398 c.G611A p.R204H Exonic 1.00 D 18 NFKBIZ Nuclear factor of kappa light polypeptide gene enhancer chr3:101576029 Splicing NA NA + - - + - Wen et al BMC Cancer 2014, 14:470 http://www.biomedcentral.com/1471-2407/14/470 Page of 11 Table Putative predispositions in familial breast cancer families and probands (Continued) 19 SMG1 SMG1 phosphatidylinositol chr16:18879624 3-kinase-related kinase c.C3083T p.T1028M Exonic 0.99 D 20 PRKCQ Protein kinase C, theta c.G855C p.Q285H 1.00 D 21 ADRA2A Adrenoceptor alpha 2A chr10:112838117 c.C363G p.C121W Exonic 1.00 D 22 PPFIA4 chr1:203025582 p.T223M Exonic 0.92 D Protein tyrosine phosphatase, receptor type chr10:6528042 c.C668T Exonic D: Probably damaging (score: 0.909-1); P: Possibly damaging (score: 0.447 - 0.908) I DNA, ATP-dependent (LIG1) in Proband #9, and Ligase IV DNA, ATP-dependent (LIG4) in Proband #10 POLQ repairs the apurinic sites [30] RAD23B plays a role in nucleotide excision repair [31] LIG1 ligates nascent DNA of the lagging strand, and a mutation in LIG1 causes replication errors, genome instability, and cancer [32] LIG4 catalyzes double-strand break repair by joining non-homologous ends, and mutation in LIG4 causes LIG4 syndrome [33] Several variants are found in wellknown oncogenes and tumor suppressor genes, such as GATA Binding Protein (GATA3) in Proband #7 and Abelson Murine Leukemia Viral Oncogene Homolog (ABL1) in Proband #18 GATA3 regulates luminal epithelial cell differentiation in the mammary gland [34,35] The abnormal expression of GATA3 causes luminal A-type breast cancer [36-38] ABL1 is a tyrosine kinase that controls cell differentiation and division It is involved in (9, 22) translocation, forming BCR-ABL fusion gene in chronic myelogenous leukemia (CML) [39] Several individual variants in different cases affect the same genes but at different positions For example, in Proband #8, a variant in KAT6B (c.G1841A/p.S614N) affects the HAT domain at the N terminal, whereas two variants in KAT6B in Family II (c.G3997T/p.D1333Y and c.C4180T/p.R1394C) affect the Met-rich domain at the C-terminal In Proband #14 and Family II, two different NOTCH2 variants (c G854A/p.R285H, c.C6178T/p.R2060C) were present Multiple variants affect the genes involved in phosphorylation These include Tyrosine Kinase Non-Receptor (TNK2) in proband #16, Phosphatidylinositol KinaseRelated Kinase (SMG1) in Proband #19, Protein Kinase C Theta (PRKCQ) in Proband #20, and Protein Tyrosine Phosphatase, Receptor Type F (PPFIZ4) in Proband #22 We also performed an analysis at the pathway level by annotating the mutation-affected genes in the three families using KEGG database (http://www.genome.jp/kegg/ pathway.html) Certain mutations were identified to affect several functional pathways For example, the genes mutated in Family I (ACADVL, AHCY, ALDOA, SGPL1, MAT1A, GALNT8, GGT1) are involved in metabolic pathways The genes mutated in Family (NOTCH2, DUSP16) are involved in Notch signaling pathway and MAPK signaling pathway; genes mutated in Family III (SLC9A1, ITGAX, ITGAD) are involved in regulation of actin cytoskeleton Discussion The majority of families with familial breast cancer lack evidence for their genetic predispositions Efforts in past decade have made slow progress in determining the unknown genetic predispositions Currently, populationbased approach is adapted as the major promising tool to reach the goal [40] One weakness of this approach is that it can “dilute out the effects of a very strong association in a small subset of the study population” [41] It requires a large-size disease population of over tens of thousands but the predispositions identified will likely remain very rare in the disease population Due to the extreme rarity, such genetic predispositions are often difficult to confirm in different disease populations and to distinguish from normal polymorphisms [5,10] Our study observed the presence of family-specific, novel, deleterious variants, and putative predispositions in the families and probands analyzed The information implies that, in addition to the population-based approach, a family-based approach provides another option to determine the genetic predisposition Based on the higher frequencies of well-known predispositions identified by traditional approaches, the rarity of the predispositions recently identified by population-based approach, and the presence of family-specific, novel, deleterious variants in disease families revealed in our study, we propose a model to explain the genetic predispositions in familial breast cancer (Figure 3) In this model, the predisposition in BRCA1 has the highest frequency in the familial breast cancer population, other known predispositions gradually decrease their frequencies to insignificant levels, and the predispositions for many BRCAx familial breast cancers are family-specific The model explains the difficulty in using traditional and population-based approaches to determine the unknown predispositions, and highlights that applying family-focused approach will be able to determine the genetic predispositions for many BRCAx disease families This model can be further tested in larger number of BRCAx familial breast cancer families Wen et al BMC Cancer 2014, 14:470 http://www.biomedcentral.com/1471-2407/14/470 Page of 11 BRCA1 TNRC9 FGFR2 BRCA2 PALB2 ATM P53 PTEN CHEK2 Figure A model for the genetic predispositions in familial breast cancer The known predisposition in BRCA1 has the highest sharing frequency in the disease population, other known predispositions decrease their frequencies towards extreme rarity in the disease populations, and the family-specific predispositions are enriched in many disease families without known predispositions The biggest circle represents the entire genetic predispositions in familial breast cancer The open circles represent the shared, known predispositions, and the black circles represent the family-specific predispositions Our study aimed to determine if there are germline mutations present, rather than reach for comprehensive coverage of germline mutations in each family We achieved this by eliminating all variants matched in population-derived variant databases (i.e., dbSNP137, ESP6500, 1000 genomes) to maximally avoid the variants representing normal polymorphism Inclusion of such variants as the predisposition candidates, even with the use of certain cut-off such as minor allele frequency (MAF)